The blood coagulation cascade involves a series of serine protease enzymes (zymogens) and protein cofactors. When required, an inactive zymogen precursor is converted into the active form, which consequently converts the next enzyme in the cascade.
The cascade is divided into three distinct segments: the intrinsic, extrinsic, and common pathways (Schenone et al., Curr Opin Hematol. 2004;11:272-7). The activation of factor X (FX) is the common point of the intrinsic and extrinsic pathways. The activation occurs either by the extrinsic complex formed by activated factor VII (FVIIa) and tissue factor, or by the intrinsic tenase complex composed of activated Factor IXa (FIXa) and activated Factor VIIIa (FVIIIa) (Mann, Thromb. Haemostasis 1999;82:165-74).
Activated FX along with phospholipids, calcium, and factor Va (FVa) then converts prothrombin to thrombin (prothrombinase complex), which in turn cleaves fibrinogen to fibrin monomers. The monomers polymerize to form fibrin strands. Factor XIIIa (FXIIIa) covalently bonds these strands to one another to form a rigid mesh.
Deficiencies of the components of the intrinsic tenase complex, FVIIIa and FIXa, lead to severe bleeding disorders, hemophilia A and B, respectively. Hemophilia A is considered the classic form of hemophilia, whereas hemophilia B is also known as Christmas disease. Hemophilia A and B are the consequence of congenital deficiencies of FVIII and FIX, respectively. The worldwide incidence of hemophilia A is approximately 1 case per 5,000 male individuals and of hemophilia B 1 case per 30,000.
Originally patients with severe hemophilia had a shortened lifespan and diminished quality of life that was greatly affected by hemophilic arthropathy. But life expectancy has increased from 11 years before the 1960s for patients who were severely affected to more than 50-60 years by the early 1980s. This has been accomplished through the widespread use of replacement therapy.
Nowadays the treatment of choice for the management of hemophilia A is replacement therapy with various plasma derived or recombinant FVIII concentrates. Although progress in the production of FVIII to ensure purity, efficacy and viral safety has been made over the past decades, some limitations remain. First of all, severe hemophilia A patients are frequently affected by anti-FVIII inhibitor antibody formation, thus rendering the therapy ineffective. Approximately 30% of patients with severe hemophilia A develop alloantibody inhibitors that can neutralize FVIII (Hay, Haemophilia 2006;12 Suppl 6:23-9, Peerlinck and Hermans, Haemophilia 2006;12:579-90). Furthermore, acquired hemophilia may occur which is the development of FVIII antibody inhibitors in persons without a history of FVIII deficiency.
Attempts to overwhelm the inhibitors with large doses of human FVIII have been tried. Also porcine FVIII which has low cross-reactivity with human FVIII antibody has been administered. More frequently, FVIII-bypassing agents, including FEIBA (factor eight inhibitor bypassing activity), FIX complex and FVIIa have also been used.
Modification of the functional activity of the tenase complex would also be an elegant approach to address several of the above discussed issues in hemophilia treatment, i.e., deficiency of FVIII or FIX and inhibitor development.
In the tenase complex FIXa has critical importance (Rawala-Sheikh et al., Biochemistry 1990;29:2606-11). FIXa is a two-chain vitamin K-dependent serine protease capable of hydrolysing the Arg194-Ile195 peptide bond in the FX molecule which leads to its activation (Venkateswarlu et al., Biophys. J. 2002;82:1190-206). Although this reaction can proceed slowly in solution, it is significantly accelerated in the presence of negatively charged phospholipid surfaces. In vivo, these surfaces are mainly provided by activated platelets and plasma lipoproteins. The rate of the reaction is increased further by the presence of FVIIIa.
FIXa exhibits very low catalytic activity in an in vitro system lacking either the co-factor FVIIIa or the physiologic substrate FX. This is in contrast to the closest related homologue, FXa, which shows significant activity towards peptide substrates (in addition to its physiologic substrate prothrombin), independent of its co-factor protein FVa. Thus the drawback of the sophisticated regulation of this enzymatic system is that failure of a single component such as FVIIIa or the development of inhibitors suffices to interrupt the functional activity of the tenase apparatus.
An improved FIX protein, which has improved FVIII-independent FX activation potential could avoid this issue. Several amino acid residues of FIXa are already known to be important for regulation of enzymatic activity and interaction with both FVIIIa and FX.
The surface loop 99 of FIXa is important for regulation of FIXa activity (Hopfner et al., Structure Fold Des. 1999;7:989-96). In the non-complexed FIXa this loop is stabilized in an inactive conformation and limits access of substrate to the catalytic machinery.
The mutations Y94F and K98T are located on the 99-loop, known to contribute to FX substrate binding by forming of the recognition site of the S2 and S4 pockets of FX. Y177F mimics the effect of activation by FVIIIa. Tyrosin 177 locks the 99-loop in an inactive conformation, which is released by binding of FVIIIa to FIXa (Sichler et al., J Biol Chem. 2003; 278:4121-26).
Val 213 and Gly 219 are conserved amino acids in most other trypsin-like proteases, and a double mutant of truncated FIX (I213V-E219G) expressed in E. coli showed increased amidolytic activity of FIXa (Hopfner et al., EMBO J. 1997;16:6626-35).
However, in none of these publications full length FIX mutants expressed in mammalian cells showed an improved functional activity in a meaningful activated partial thromboplastin time (aPTT) assay in FVIII-depleted plasma or FVIII-inhibitor-patient plasma.
Thus, there remains a great need in the art for compositions and methods that provide an improved FIX molecule that can be used for treatment of patients with hemophilia A.
It was the inventive task of the present invention to develop novel FIX proteins by introduction of amino acid exchanges, which have improved FVIII-independent FX activation potential with coagulation FVIII activity useful for the treatment of bleeding disorders.